技术文档
丙酮、TCA和甲醇等有机试剂蛋白质浓缩方法步骤
Precipitation techniques
Purpose: to concentrate the sample and remove small interfering species, such as salts & detergents, fordownstream applications e.g. 2D gels.For small amounts of protein (< low microgram) it may be necessary to add a carrier protein such asinsulin.
Acetone Precipitation Protocol
1. Cool the required volume of acetone to -20°C.
2. Place protein sample in acetone-compatible tube.
3. Add four times the sample volume of cold (-20°C) acetone to the tube.
4. Vortex tube and incubate for 60 minutes at -20°C.
5. Centrifuge 10 minutes at 13,000-15,000 x g.
6. Decant and properly dispose of the supernatant, being careful to not dislodge the protein pellet.
Optional: If additional cycles of precipitation are necessary to completely remove the interfering
substance, then repeat steps 2-5 before proceeding to step 7.
7. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. Do
not over-dry pellet, or it may not dissolve properly.
8. Resuspend in appropriate buffer.
TCA Precipitation Protocol
1. Add an equal volume of 20% TCA (trichloroacetic acid) to protein sample.
2. Incubate 30 min on ice.
3. Spin in microfuge at 4 deg. For 15 min.
4. Carefully remove all supernatant.
5. Add ~300 ul cold acetone and spin 5 min at 4 degrees.
6. Remove supernatant and dry pellet.
7. Resuspend samples in desired buffer.
Warning: TCA is a strong acid and should be handled with care
Chloroform/Methanol Precipitation
1. To sample of starting volume 100 ul
2. Add 400 ul methanol
3. Vortex well
4. Add 100 ul chloroform
5. Vortex
6. Add 300 ul H2O
7. Vortex
8. Spin 1 minute @ 14,0000 g
9. Remove top aqueous layer (protein is between layers)
10. Add 400 ul methanol
11. Vortex
12. Spin 2 minutes @ 14,000 g
13. Remove as much MeOH as possible without disturbing pellet
14. Speed-Vac to dryness
15. Bring up in 2X sample buffer for PAGE
Reference: Wessel, D. and Flugge, U. I. Anal. Biochem. (1984) 138, 141-143